Tacrolimus (also known as ProGraf® and FK506) is a macrolide lactone antibiotic with potent immunosuppresive properties isolated from the soil fungus Streptomyces tsukubaensis. Tacrolimus exhibits its immunosuppressive effect by inhibiting the calcineurin pathway (which ultimately inhibits T-cell proliferation) through the formation of a pentameric complex between tacrolimus, FK binding protein (FKBP), calmodulin, and calcineurins A and B (McKeon et al., Cell Vol. 66, pages 823-6 (1991)). Tacrolimus binds tightly to FKBP (Kd˜0.4 nM), and is also known to bind to cyclophilin (Handschumacher et al., Science Vol. 226, pages 544-547 (1984)), albumins, and alpha-1-acid glycoprotein (Wong, Clinica Chimica Acta Vol. 313, pages 241-253 (2001). Tacrolimus, used in combination with other immunosuppressants, has gained broad acceptance for the treatment of tissue rejection following organ transplantation.
Extreme inter-patient variability between drug dosage and drug blood levels leads to its potential for toxicity, making therapeutic monitoring of tacrolimus levels in patients undergoing tacrolimus immunosuppressive therapy a standard practice.
Tacrolimus is extensively sequestered in erythrocytes (red blood cells or RBCs), bound to FK binding protein (FKBP; therefore, whole blood samples are a preferred matrix for therapeutic blood monitoring of tacrolimus, even if it can be extracted form biopsies samples, bone marrow and other body fluids. Tacrolimus also forms complexes with other blood plasma constituents such as immunophilins, albumin and lipoproteins. In order to determine the presence of tacrolimus and its concentration in a human blood sample, RBC's present in the sample must be lysed to release the tacrolimus/protein complexes. Then the tacrolimus/protein complexes must be dissociate to release tacrolimus.
Tacrolimus has limited solubility in aqueous solutions. Consequently, all available tacrolimus assays use organic solvents to extract the drug for assay. Organic solvents (eg., ethyl-acetate, methanol, methylene chloride) have been used routinely to lyse the red blood cells, to denature proteins and to extract tacrolimus in preparation for its quantification.
The use of organic solvent-based procedures suffer form several significant disadvantages. For example, organic solvents are highly volatile, highly flammable, and involve hazardous materials that must be properly disposed of in accordance with environmental guidelines.
The volatility of organic solvents can also interfere with accurate quantification of tacrolimus on account of the accumulative evaporation of the volatile solvents. Excessive evaporation at each step of extraction, and during sample assay incubations, can result in the detection of artificially elevated levels of tacrolimus.
All current procedures for tacrolimus extraction require a complex manual sample preparation. Current procedures require a sample denaturation/extraction step, a centrifugation step and a supernatant decantation step. Currently used manual tacrolimus extraction procedures are slow, labor intensive processes. They typically involve precision pipetting of four components: the sample, the denaturant agent, the extraction agent and the extracted sample. Manual precision pipetting is time consuming and potentially unreliable. Consequently these procedures are expensive in terms of the costs associated with staffing. They are also technically limited in terms of analytical throughput and tacrolimus quantification accuracy.
The use of organic solvents along with denaturation, centrifugation, and decantation steps, required with current sample pretreatment procedures preclude the full automation of tacrolimus quantification assay. For full automation of quantification to be possible, all of the reagents and samples must remain in an aqueous form. Denaturation (lysis) and centrifugation of the blood sample would no longer be required. The use of aqueous solutions would also eliminate the excessive evaporation effects, and the risks associated with the use of volatile, flammable, potentially explosive conditions in an electrical device.
Accordingly, there is a need for and interest in the art of devising a fully automated method for tacrolimus quantification that eliminates the need for elaborate and cumbersome sample pretreatment. Such a method would represent a considerable improvement over current methods known in the art in terms of cost per test. The elimination of the manual interaction of the user with the samples would also improve method reproducibility by eliminating human errors, which can affect the drug extraction (and hence, the subsequent result).
The present invention provides a method of extracting tacrolimus in a non-precipitating, non-denaturing aqueous environment, which not only eliminates the use of organic solvents and centrifugation, but also fulfills the requirements for full automation of tacrolimus quantification assays.